High performance liquid chromatography (HPLC) is a process by which one or more compounds from a chemical mixture may be separated and identified. A transport liquid, for example a solvent, is pumped under high pressure through a column of packing medium, and a sample of the chemical mixture to be analyzed is injected into the column. As the sample passes through the column with the liquid, the different compounds, each one having a different affinity for the packing medium, move through the column at different speeds. Those compounds having greater affinity for the packing material move more slowly through the column than those having less affinity, and this speed differential results in the compounds being separated from one another as they pass through the column.
The transport liquid with the separated compounds exits the column and passes through a detector, which identifies the molecules, for example by spectrophotometric absorbance measurements. A two dimensional plot of the detector measurements against elution time or volume, known as a chromatogram, may be made, and from the chromatogram the compounds may be identified.
For each compound, the chromatogram displays a separate curve or “peak”. Effective separation of the compounds by the column is advantageous because it provides for measurements yielding well defined peaks having sharp maxima inflection points and narrow base widths, allowing excellent resolution and reliable identification of the mixture constituents. Broad peaks, caused by poor column performance, are undesirable as they may allow minor components of the mixture to be masked by major components and go unidentified.
Columns for HPLC typically comprise high strength stainless steel tubes packed under high pressure with packing media comprising, for example, silane derivatized silica spheres having a diameter less than 20 microns. The packing media is held within the tube by sintered stainless steel frits. The frits are porous and allow the transport liquid and the sample to pass through the tube. It is advantageous to press the frits into the bore of the tube with an interference fit to prevent the packing material from escaping past the frits. Threaded end fittings are positioned at opposite ends of the tube. The end fittings keep the frits within the bore, compress the packing media to help maintain its hydraulic orientation, and are adapted to receive standard fittings from capillary tubes for connecting the column to a chromatograph.
Dead space within a column should be avoided because it allows mixing of the transport liquid and the sample that degrades the column performance. This manifests itself as a broadening of the peaks and a concomitant decrease the resolving capability of the HPLC apparatus.